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NUCLEIC ACIDS
DNA(Deoxyribonucleic acid)
DNA
RNA (Ribonucleic acid)
Brief History of Nucleotides
and Nucleic acids
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1869 – Johannes Friedrich Miescher isolated
“nuclein” from soiled bandages.
1902 – Archibald Garrod studied “Alkaptonuria”;
he also concluded that specific gene is associated
with absence of specific enzyme.
1903 – Walter Sutton, introduced the
chromosome structure.
1913 – Thomas Hunt Morgan “gene mapping”
1926 – James Sumner purified urease and he also
identified enzymes to be proteins.
Brief History of Nucleotides
and Nucleic acids
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1928 –Frederick Griffith “Transforming
Principle” a chemical transferred from dead
bacteria to living cells caused genetically
converted strains.
1944 – Oswald Avery, Maclyn McCarty, and
Colin MacLeod – identified Griffith’s
“transformation principle” as DNA.
1947 - Erwin Chargaff – studied the base
pairing
1950’s – Rosalind Franklin “X-ray of DNA”
Brief History of Nucleotides
and Nucleic acids
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1953: James Watsons and
Francis Crick – “DNA double
helix”.
"Molecular structure of
Nucleic Acids: A Structure for
Deoxyribose Nucleic Acid"
An article published in Nature
(April 25, 1953).
It was the first publication
which described the discovery
of the double helix structure
of DNA.
Watson & Crick proposed...
• DNA had specific pairing
between the nitrogen
bases:
“Rungs of ladder”
Nitrogenous
Base (A,T,G or C)
• ADENINE – THYMINE
• CYTOSINE - GUANINE
• DNA was made of 2 long
stands of nucleotides
arranged in a specific way
called the
“Complementary Rule”.
DNA double helix
“Legs of ladder”
Phosphate &
Sugar Backbone
BASE-PAIRINGS
H-bonds
G
C
T
A
THE WATSON-CRICK MODEL
BASE-PAIRING IN DNA
The two strands of DNA are arranged antiparallel to one another:
viewed from left to right the "top" strand is aligned 5' to 3', while the
"bottom" strand is aligned 3' to 5'.
Nucleotides and Nucleic acids
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are biological molecules
that possess heterocyclic
nitrogenous bases as
principal components of
their structure.
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The biochemical roles of
nucleotides: participate as essential
intermediates in virtually all
aspects of cellular metabolism.
Serving an even more central
biological purpose are the nucleic
acids, the elements of heredity and
the agents of genetic information
transfer.
Just as proteins are linear polymers
of amino acids, _________ are
linear polymers of nucleotides.
Nucleic acids are “Polymers”
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Responsible for the storage and
passage of the information needed
for the production of proteins;
The chemical link between
generations;
The source of genetic information in
chromosomes;
Dictate amino-acid sequence in
proteins; and
Give information to chromosomes,
which is then passed from parent to
offspring.
Nitrogenous Bases
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The bases of nucleotides and nucleic acids
are derivatives of either
pyrimidine or purine.
Q: Look at the structure of pyrimidine,
what can you observe?
A: 6 member heterocyclic ring, with 2
Nitrogen atoms and it is number
clockwise.
Q: Look at the structure of purine, what
can you observe?
A: It is 9 member ring structure of
pyrimidine fused with an imidazole ring.
Common Pyrimidines and
Purines
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Q: What are the common
pyrimidine bases?
A: Cytosine, Uracil and Thymine
Q: What is the name of the first
pyrimidine base? What is its
chemical name?
Q: What is the name of the
second pyrimidine base? What is
its chemical name?
Q: What is the name of the third
pyrimidine base? What is its
chemical name?
1.
2.
3.
Common Pyrimidines and
Purines
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Q: What are the common
purine bases?
A: Adenine and Guanine
Q: What is the name of
the first purine base?
What is its chemical
name?
Q: What is the name of
1.
the first purine base?
What is its chemical
name?
2.
Properties of Pyrimidines and
Purines

keto-enol tautomeric
shifts
 pyrimidines
and purines
exist as tautomeric pairs

strong absorbance of
ultraviolet (UV) light,
which is also a
consequence of the
aromaticity of their
heterocyclic ring
structures.
Properties of Pyrimidines and
Purines

Hydrogen bonding
between purine and
pyrimidine bases is
fundamental to the
biological functions of
nucleic acids, as in the
formation of the double
helix structure of DNA.
Properties of Pyrimidines and
Purines

The important functional groups
participating in H-bond formation:
 amino
groups of adenine, cytosine
and guanine;
 the ring nitrogens at position 3 of
pyrimidines and position 1 of purines;
and
 the strongly electronegative oxygen
atoms attached at position 4 of uracil
and thymine, position 2 of cytosine,
and position 6 of guanine
Pentose Sugar
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Five-carbon sugars
are called _____.
Q: What is the
pentose sugar
present in DNA?
Q: What is the
pentose sugar
present in RNA?
Pentose Sugar
Pentose is in the fivemembered ring form
known as _______.
 Q: What is the furanose
present in DNA?
 A: 2-deoxy-Dribofuranose
 Q: What is the furanose
present in RNA?
 A: D-ribofuranose

Nucleosides
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Nucleosides are compounds
formed when a base is linked
to a sugar via a glycosidic bond.
A glycosidic bond is a type
of covalent bond that joins a
sugar molecule to another group.
Q: What is the name of the
nucleoside if the sugar found is
ribose?
Q: What is the name of the
nucleoside if the sugar found is 2deoxyribose?
+
Nucleosides
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In nucleosides, the
bond is an N-glycoside
because it connects the
anomeric C-1' to N-1 of
a pyrimidine or to N-9
of a purine.
Nucleoside Nomenclature
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Nucleosides are named by adding the ending -idine to the
root name of a pyrimidine or -osine to the root name of a
purine.
Nitrogenous base
Cytosine + sugar
Guanine + sugar
Adenine + sugar
Uracil + sugar
Thymine + sugar
Nucleoside name
Cytidine
Guanosine
Adenosine
Uridine
Thymidine
Nucleoside Conformation
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syn and anti
rotation of the base about the glycosidic bond is
sterically hindered, principally by the hydrogen atom on
the C-2' carbon of the furanose
Pyrimidine nucleosides favor the anti conformation
Purine nucleosides can adopt either the syn or anti
conformation.
Nucleosides Are More WaterSoluble Than Free Bases
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Nucleosides are much more water-soluble than
the free bases because of the hydrophilicity of the
sugar moiety.
Nucleosides are relatively stable in alkali.
Pyrimidine nucleosides are also resistant to acid
hydrolysis, but purine nucleosides are easily
hydrolyzed in acid to yield the free base and
pentose.
Nucleotides Are Nucleoside
Phosphates
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The nucleoside ribose ring
has three —OH groups
available for esterification,
at C-2', C-3', and C5'(although 2'-deoxyribose
has only two). The vast
majority of monomeric
nucleotides in the cell are
ribonucleotides having 5'phosphate groups.
A nucleotide results
when phosphoric acid is
esterified to a sugar —
OH group of a
nucleoside.
Nomenclature of Nucleotide
Q: What are the names of
this structure?
Q: What are the names of
this structure?
Answers:
Answers:
1.) adenosine 5’-monophosphate 1.) guanosine 5’-monophosphate
2.) AMP or 5’-AMP
2.) GMP or 5’-GMP
3.) adenylic acid
3.) guanylic acid
4.) adenylate
4.) guanylate
Nomenclature of Nucleotide
Q: What are the names of
this structure?
Q: What are the names of
this structure?
Answers:
1.) cytidine 5’-monophosphate
2.) CMP
3.) cytidylic acid
4.) cytidylate
Answers:
1.) uridine 5’-monophosphate
2.) UMP
3.) uridylic acid
4.) uridylate
Cyclic Nucleotides
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Nucleoside monophosphates in
which the phosphoric acid is
esterified to two of the available
ribose hydroxyl groups are found in
all cells.
Forming two such ester linkages with
one phosphate results in a cyclic
structure.3',5'-cyclic AMP, often
abbreviated cAMP, and its guanine
analog 3',5'-cyclic
GMP, or cGMP, are important
regulators of cellular metabolism.
Nucleoside Diphosphates and
Triphosphates
Nucleoside 5'-Triphosphates Are
Carriers of Chemical Energy
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ATP has been termed the energy currency of the
cell.
GTP is the major energy source for protein
synthesis.
CTP is an essential metabolite in phospholipid
synthesis
UTP forms activated intermediates with sugars
that go on to serve as substrates in the
biosynthesis of complex carbohydrates and
polysaccharides.
Nucleoside 5'-Triphosphates Are
Carriers of Chemical Energy
Nucleoside 5'-Triphosphates Are
Carriers of Chemical Energy
The evolution of metabolism has led to the
dedication of one of these four NTPs to
each of the major branches of metabolism.
 To complete the picture, the four NTPs and
their dNTP counterparts are the substrates
for the synthesis of the remaining great
class of biomolecules—the nucleic acids.
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The Bases of Nucleotides Serve as
“Information Symbols”
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ATP: serve as the primary nucleotide in central pathways
of energy metabolism,
GTP: is used to drive protein synthesis.
Various nucleotides are channeled in appropriate
metabolic directions through specific recognition of the
base of the nucleotide.
This role as information symbols extends to nucleotide
polymers, the nucleic acids, where the bases serve as the
information symbols for the code of genetic information.
Nucleic Acids Are
Polynucleotides
Activity
1. Identify the components of this structure.
2. Classify the structure as that of a nucleoside or
a nucleotide.
3. Identify the nitrogenous whether a purine or
pyrimidine.
4. What is the sugar component in the structure?
5. What is the name of the type of bond that
occurs between a phosphate and ribose
group?
6. Between a ribose and nitrogen base group?
7. Name the bond that forms between
nucleotide groups.
8. Explain the significance of hydrogen bonds in
DNA helices.
Solution to the Activity
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The structure contains a
nitrogenous base and a pentose.
It is a nucleoside, because it
contains a pentose and a
nitrogenous base.
The nitrogenous base is
pyrimidine. Cytosine.
The sugar has a -H in the 2'
position and is therefore
deoxyribose.
Solution to the Activity
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An ester bond forms between a phosphate group and a
ribose group.
A glycosidic bond forms between a ribose group and a
nitrogen base group
A phosphodiester bond forms between nucleotides to
form nucleic acids.
While hydrogen bonds do contribute a small amount to
the stability of helices, their main contribution is to the
specificity of a helix. Hydrogen bonds dictate the
complementary base pairing that aligns anti-parallel
nucleic acids strands in a DNA helix.
Chemical Nature of DNA
Chemical Nature of DNA
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3.
DNA at a glance...
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DNA is organized into chromosomes,
which are found within the nuclei of
cells.
A gene is a segment of DNA on a
chromosome that codes for specific
protein and thus determines a trait.
The genetic code is determined by the
order of bases in the gene, which
specifies what type of proteins will be
produced.
Remember this! DNA contains the
genetic material.
DNA – Deoxyribonucleic acid
Chemical basis of heredity
and is organized into genes,
the fundamental units of
genetic information.
FUNCTIONS:
1.
Involve in replication during
cell division
2.
Gene expression by
transcription
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DNA – Deoxyribonucleic acid
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In eukaryotic cells, it is
present in chromosomes
in the nucleus
It is also present in
mitochondria and in the
chloroplast of plants
Prokaryotic cells, which
lack nuclei, have a single
chromosome but may
also contain
nonchromosomal DNA in
the form of plasmid.
Structure of DNA
Structure of DNA
Polydeoxyribonucleotide
 Linked by 3’,5’-phosphodiester bonds
 Double stranded molecules
 In eukaryotic cells: nucleoproteins
 In prokaryotic cells: nucleoid
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Structure of DNA
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3’,5’-phosphodiester bonds
Structure of DNA
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3’,5’-phosphodiester bonds
where the
phosphate group
of the next
nucleotide in a
series can be
linked
where the
phosphate group of
the previous
nucleotide is linked
Structure of DNA
Structure of DNA:
Double Helix
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Common axis
The chains are paired in anti-parallel manner
Structure of DNA:
Double Helix
“Rungs of ladder”
Nitrogenous
Base (A,T,G or C)
“Legs of ladder”
Phosphate &
Sugar Backbone
Structure of DNA:
Base Pairing
Note: the hydrogen
bonds plus the
hydrophobic
interactions between
the stacked bases
stabilize the structure
of the double helix.
Separation of the two DNA
strands in the double helix
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Q: How the separation occurs?
A: The 2 strands of the double helix occur when
the hydrogen bonds between the paired bases are
disrupted.
Q: What is the mechanism of disruption?
A: The pH of the DNA solution is altered so that
the nucleotide bases ionize or if the solution is
heated.
Note: no phosphodiester bond bonds are
broken by such treatment.
Separation of the two DNA
strands in the double helix
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Remember these terms:
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Melting Temperature (Tm): the temperature at which the half
of the helical structure is lost.
DNA denaturation: loss of helical structure of the DNA by
increasing the temperature(melting) or treatment with alkali.
Hyperchromic effect: DNA absorbance to UV is at 260
nm(max), it will increase if the DNA dissociate.
Renaturation(Re-annealing): occurs when the complementary
DNA strands can reform the double helix .
Hypochromic effect: absorbance at 260 nm decreases.
Note: DNA saturated with Adenine-Thymine denature at
low temperature.
Structural Forms of DNA
•
There are at least 6
structural forms:
A – E and Z
Structure of B-DNA
•
Q: What is the
common form of DNA?
•
A: B-form is the most
common; found under
physiologic condition
Structural Forms of DNA
Structural Forms of DNA
Width of
double helix
is 20 A
One complete turn
is 34 A containing
10 base pairs, so the
rise is 3.4 A
Structure of B-DNA
Structural Forms of DNA
B-DNA: right-handed
helix wi th 10 residues
per 360° turn of the
helix. Mostly found in
DNA chromosomes.
A-DNA: produced by
moderately dehydrating BDNA, right-handed helix with
11 residues per turn and tilted
by 20° away from the
perpendicular axis
Z-DNA: left
handed helix
with 12 residues
per turn.
Modifications of DNA
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Can be modified by translation process(after DNA
synthesis).
Most common: methylation of cytosine that precedes
guanosines to produce methyl-cytosine.
Frequency of methylation: <10% of all cytosines.
Function of methylation: the presence of methylcytosines
in genes is strongly correlated with transcriptionally
inactive genes.
Note: Methylcytosine in sequence of alternating CG
doublets favors the transition of DNA from the B-form to
Z-form
PLASMIDS
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 A small, circular,
extrachromosomal DNA
molecules
Plasmid DNA carries genetic
A prokaryotes contains a
information and undergoes
single, double-stranded,
replication that may or may
super-coiled circular
not be synchronized to
chromosomes.
chromosomal division
Each chromosome is
Used as vectors in
associated with histonerecombinant DNA
like proteins and RNA that
technology
can condense the DNA to
form a nucleoid.
Names of DNA Base
Derivatives
Base
Nucleoside
5'-Nucleotide
Adenine 2'-Deoxyadenosine 2'-Deoxyadenosine-5'-monophosphate
Cytosine 2'-Deoxycytidine
2'-Deoxycytidine-5'-monophosphate
Guanine 2'-Deoxyguanosine 2'-Deoxyguanosine-5'-monophosphate
Thymine 2'-Deoxythymidine 2'-Deoxythymidine-5'-monophosphate
Van der waals
and
hydrophobic
interactions
between the
stacked
adjacent base
pairs
The phosphodiester linkage of monomer units into a
polymeric molecule is called the PRIMARY STRUCTURE
The Primary Structure
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The arrangement (or order) of specific
nucleotides along the chain is called the
sequence.
The sequence is genetic information.
A sequence can be written simply as: ACGTT (or
5'-ACGTT-3').
Alternatively, the sequence could also be written
as: 3'-TTGCA-5'.
Higher Order Structure
of DNA
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I. Supercoils: studies of circular DNA have
shown that it can be twisted into compact
supercoiled or superhelical form.
 Right-handed(positive)
supercoil: are twisted in the
same direction as the right-handed helix of B-form
DNA about its axis.
 Left-handed(negative) supercoil: twisted on the
opposite direction.
Higher Order Structure
of DNA
Topoisomerases: enzymes responsible for
altering the superhelicity of the cellular
DNA. This enzyme is very important in the
process of replication.
 Note: Supercoils have a high free energy
compared to relaxed DNA.
 Supercoil exist naturally, examples,
bacterial genomic DNA and
plasmid(mostly are negative).

Higher Order
Structure of DNA
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II. Chromatin: In humans, DNA is
divided and packaged into 46 separate
structures known as chromosomes.
During the interphase, when the DNA
needs to be accessible to the
transcription and replication enzymes,
it is packaged into less densely mitotic
chromosomes known as chromatin.
Chromatin can be detected by
microscopic staining, the DNA is
packaged with approximately double
its mass of protein.
Higher Order
Structure of DNA
Histones: the major class of proteins
associated with chromatin which exist
in a ass approximately equal to DNA in
the chromatin.
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Small (11,000 – 21,000 MW), basic proteins that bind to the
acidic DNA by noncovalent interactions to form nucleosomes.
Four core histones: H2a, H2b, H3 and H4 an one linker
histomes: H1
In many eukaryotes there are amino acid sequence variants of
all the histones except H4.
All histones are post-translationally modified at various stages
of cell cycle.
DNA SYNTHESIS
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View:
1.
How Nucleotides are added in DNA
2.
DNA replication fork
QUIZ
1. DNA polymerase III can only add nucleotides to an
existing chain, so _________________ is required.
A) an RNA primer
B) DNA polymerase I
C) helicase
D) a DNA primer
2. The enzymes that break hydrogen bonds and unwind
DNA are:
A) Primers
B) Forks
C) Helicases
D) Polymerases
QUIZ
3. Replication begins at a specific site in the DNA
called ___________.
4. During replication within the fork,________ bind
to the single-stranded regions preventing the
strands from rejoining.
5. This enzyme replaces the primase and is able to
add DNA nucleotides to the RNA primer
6. Short fragment or sequences of discontinuous
DNA
QUIZ
7. DNA replication is from 5’ to 3’ direction. True or
False.
8. -9. In which strand DNA is synthesized
continuously? Discontinuously?
10. The DNA fragments on the lagging strand are
hooked together by the enzyme________.
DNA REPLICATION
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Primary function is understood
to be the provision of progeny
with the genetic information
possessed by the parent.
Replication must be complete
and carried out in such a way
as to maintain genetic stability
within the organism and the
species.
The process of DNA replication
is complex and involves many
cellular functions and several
verification procedures to
ensure fidelity in replication.
Is DNA replication conservative or
semiconservative?
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Conservative - old strand acts as a template
 One daughter
strand is the original template
while the other strand is composed entirely out
of new nucleotides
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Semiconservative - old strand splits apart
and acts as a template
 Both
daughter strands are composed of one of
the old strands and one comprised out of new
nucleotides
Is DNA replication conservative or
semiconservative?

In their brief paper, "Molecular structure of
Nucleic Acids: A Structure for Deoxyribose
Nucleic Acid" , James Watson and Francis Crick
(1953) wrote:
 "It
has not escaped our notice that the specific pairing
we have postulated immediately suggests a possible
copying mechanism for the genetic material."

This mechanism for DNA replication is known as
the semiconservative model.
Is DNA replication conservative or
semiconservative?
Semi-Conservative Model
The two parental strands
separate and each makes a copy
of itself.
After one round of replication,
the two daughter molecules each
comprises one old and one new
strand.
Note that after two rounds, two
of the DNA molecules consist
only of new material, while the
other two contain one old and
one new strand.
Conservative Model
The parental molecule directs
synthesis of an entirely new
double-stranded molecule, such
that after one round of
replication, one molecule is
conserved as two old strands.
This is repeated in the second
round.
Dispersive Model
Material in the two parental
strands is distributed more or
less randomly between two
daughter molecules.
In the model shown here, old
material is distributed
symmetrically between the two
daughters molecules.
Other distributions are
possible.
DNA Replication by
Complementary Base Pairing
DNA Replication by
Complementary Base Pairing
Steps involved in DNA
Replication in Eukaryotes
Identification of origin of replication
Unwinding (denaturation) of dsDNA to provide
a ssDNA template
Formation of the replication fork
Initiation of DNA synthesis and elongation
Formation of replication bubbles with ligation
of the newly synthesized DNA segments
Reconstitution of chromatin structure
1.
2.
3.
4.
5.
6.
•
•
E.coli is used to describe the replication process in prokaryotes
Less complex in prokaryotes
STEP 1: Identification of
origin of replication
Origin of replication in
eukaryotic DNA
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Multiple sites
 Provides
mechanism for rapidly replicating the
great length of the eukaryotic DNA

These sites include a short sequence
composed almost exclusively of A-T base
pairs called CONSENSUS SEQUENCE
 The
order of nucleotide is essentially same at
each site
Replication begins at a specific site in the DNA called
the origin of replication
Eukaryotic DNA – multiple origin of replication
Origin of replication in the prokaryotic circular DNA
STEP 2: Unwinding (denaturation) of
dsDNA to provide a ssDNA template
DNA Replication by Complementary Base
Pairing: Unwinding by DNA Helicase
STEP 3: FORMATION OF THE
REPLICATION FORKS
DNA is
synthesized
continuously
DNA is
synthesized
discontinuously
• Replication forks
are the actual site of
DNA copying
•During replication
within the fork,
helix destabilizing
proteins bind to the
single-stranded
regions preventing
the strands from
rejoining.
Proteins required for DNA separation
to form the Replication Fork

DnaA protein
 Bind
to specific nucleotide sequences at the ORIC
 Causes the double-stranded DNA to melt
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Strands separate, forming localized regions of single-stranded
Single-stranded DNA-binding (SSB) proteins
 Also
called helix-destabilizing proteins
 Keep 2 strands of DNA separated in the area of
replication origin
 Also protect DNA from nucleases that cleave ssDNA

DNA helicases
 Bind
to ssDNA near the replication fork
 Unwind the double helix
 Requires energy provided by ATP
STEP 4: Initiation of DNA synthesis
and Elongation
RNA PRIMER
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A short, doubled stranded
region with a free OHgroup on the 3’ end of the
shorter strand
Synthesized by primase
(specific RNA polymerase)
Required by DNA
polymerase to initiate
synthesis of a
complementary strand.
STEP 4: Initiation of DNA synthesis
and Elongation
DNA Polymerases
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Responsible for copying the DNA templates
Only able to “read” in the 3’  5’ direction and synthesize
new DNA in the 5’ 3’ direction
Elongate a new DNA strand by adding
deoxyribonucleotide, one at a time, to the 3’-end of the
growing chain
DNA POLYMERASE I
Replaces the primase and is able
to add DNA nucleotides to the
RNA primer
 Catalyze DNA chain elongation

5. Formation of replication bubbles with
ligation of the newly synthesized DNA
segments
1
1
1
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DNA polymerase I digests away the RNA primer
and replaces the RNA nucleotides of the primer
with the proper DNA nucleotides to fill the gap
Finally, the DNA
fragments on the
lagging strand are
hooked together by
the enzyme
DNA ligase
Okazaki Fragments
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Short fragment or sequences of discontinuous
DNA
Eventually joined to become a single continuous
strand
Classes of Proteins involved in
Replication
PROTEIN
FUNCTION
DNA polymerases
Deoxynucleotide polymerization
Helicases
Processive unwinding of DNA
Topoisomerases
Relieve torsional strain that results from
helicase-induced unwinding
Primase
Initiates synthesis of RNA primers
Single-strand binding
proteins
Prevent premature reannealing of dsDNA
DNA ligase
Seals the single strand nick between the nascent
chain and Okazaki fragments on lagging strand
DNA Topoisomerase I
 Solves the problem caused by tension generated by
winding/unwinding of DNA.
 It wraps around DNA and makes a
cut (producing a “nick”) permitting
the helix to spin.
 Once DNA is relaxed,
topoisomerase reconnects broken
strands
 Relax negative supercoils in E.coli,
and both negative and positive
supercoils in eukaryotic cells
Note: Have both strand-cutting (nuclease) and strandresealing (ligase) activities
DNA topoisomerase II
– makes transient breaks in both
strands
cut both strands of the DNA helix simultaneously in order to
manage DNA tangles and supercoils.
In this process, these enzymes change the linking number of
circular DNA by +/-2
The DNA Polymerases
Three (3) important properties
1. Chain elongation
2. Processivity
3. Proofreading – ensures replication
fidelity
Prokaryotic and Eukaryotic
DNA polymerases
E.coli Mammalian
Function
I

Gap filling and synthesis of
lagging strand
II

DNA proofreading and repair

DNA repair

Mitochondrial DNA synthesis

Processive, leading strand
synthesis
III
DNA Polymerases
Enzyme
Activity
DNA Polymerase
I
DNA
DNA
Polymerase
Polymerase
II
III
5' to 3'
polymerase
Yes
Yes
Yes
3' to 5'
exonuclease
Yes
Yes
Yes
5' to 3'
exonuclease
Yes
No
No
E. coli DNA Polymerase I

5'-to-3' DNA Polymerase activity



Gap is filled
3'-to-5' exonuclease (Proofreading activity)
5'-to-3' exonuclease (Nick translation activity)


Remove one nucleotide at a time from region of
DNA that is properly base-paired
Also remove groups of altered nucleotides in the
5’→3’ direction, removing from 1 to 10 nucleotides
at a time
5’ to 3’ Exonuclease Activity

DNA polymerase I
uses its 5' to 3'
exonuclease activity
to digest away the
primer RNA, and
replaces the primer
with DNA by
extending the strand
from the adjacent
Okazaki fragment
DNA polymerase III – 3’ to 5’
exonuclease activity
Excision of mismatched nucleotides by a 3' exonuclease site
distant from the polymerase site
3' to 5' Exonuclease Action
ENDONUCLEASE
EXONUCLEASE
Cleave within the chain
to produce singlestranded nicks
Cleave from the end of
the chain, releasing
single nucleotide
5’
3’
3’
A
C
G
A
T
C
A
T
G
C
T
A
G
T
5’
dRAp
Eukaryotic DNA Replication
Polymerases are designated by Greek letter
 Pol  - has primase activity
 Pol  - elongate the leading strand
 Pol  - elongate the lagging strand in mammals
 Pol  and Pol


 both have 3’ to 5’ exonuclease activity
Pol  - analogous to E.coli DNA polymerase—that
is, it can, in association with other proteins,
excise primers and carry out “repairs”
Pol  - replicates mitochondrial DNA
STRUCTURAL ORGANIZATION OF EUKARYOTIC DNA
Organization of Eukaryotic DNA
HISTONES
 Basic proteins tightly
bound to eukaryotic
DNA
 5 classes: H1, H2A, H2B,
H3, and H4
 H1 binds to the DNA
chain between the
nucleosome beads
 H1 appears to aid the
packing of nucleosomes
into the more structures
NUCLEOSOMES
 Nucleosomes “beads” is
formed by two molecules
each of H2A, H2B, H3,
and H4 with DNA double
helix wound around
nearly twice
 Nucleosomes, joined by
“linker” DNA (approx. 50
nucleotides in length)
form a polynucleosome
or nucleofilament.
Stage Activity
Duration
G1
Growth and
increase in cell
size
10 hr
S
DNA synthesis
8 hr
G2
Post-DNA
synthesis
5 hr
M
Mitosis
1 hr
Cell cycle for a hypothetical cell with
a 24 hr cycle
DNA DAMAGE
CAUSES OF DNA DAMAGE
1.
UV LIGHT - RADIATION
2.
CHEMICALS
3.
OTHER AGENTS
DNA DAMAGE CAN BE:
1.
BASE ALTERATION
2.
REMOVAL OR LOST OF NUCLEOTIDE BASE
Four Mechanisms of DNA Repair
MECHANISM
PROBLEM
SOLUTION
MISMATCH REPAIR
Copying errors
Methyl-directed strand
cutting, exonuclease
digestion, and
replacement
BASE EXCISION REPAIR
Spontaneous, chemical,
or radiation damage to a
single base
Base removal by Nglycosylase, abasic sugar
removal, replacement
NUCLEOTIDE EXCISION Spontaneous, chemical,
REPAIR
or radiation damage to a
DNA segment
Removal of an approx. 30nucleotide oligomer and
replacement
DOUBLE-STRAND
BREAK REPAIR
Synapsis, unwinding,
alignment, ligation
Ionizing radiation,
chemotherapy, oxidative
free radicals
Proteins involved in the DNA
repairing of E. coli
Mismatch Repair
DNA Repair by Base Excision
DNA repair by Nucleotide Excision
Exinuclease cuts the defective region
Double Strand Break Repair
IF DNA DAMAGE IS NOT
REPAIRED
If DNA damage is
too extensive to
repair
PERMANENT MUTATION
Loss of control over the
proliferation of mutated cell,
leading to cancer
Cells undergo
apoptosis
(programmed
cell death)
Diseases resulting from disordered
DNA repair capabilities

Xeroderma Pigmentosum (XP)
 XP is a rare inherited disease
of humans which, among other
things, predisposes the patient
to pigmented lesions on areas
of the skin exposed to the sun
and an elevated incidence of
skin cancer.
 XP can be caused by mutations
in any one of several genes —
all of which have roles to play
in NER
Diseases resulting from disordered
DNA repair capabilities
FANCONI’S ANEMIA
• An inherited disease that mainly affects the
bone marrow.
• The disease is caused by a genetic defect.
• This defect prevents cells from fixing
damaged DNA or removing toxic
substances called oxygen-free radicals that
damage cells.
• People with certain birth defects or who
develop low blood counts may have this
disease.
Diseases resulting from disordered
DNA repair capabilities
ATAXIA-TELANGIECTASIA



Rare, inherited, childhood
disease that affects the brain and
other parts of the body.
Ataxia refers to uncoordinated
movements, such as walking.
Telangiectasis is the
enlargement of blood vessels
(capillaries) just below the
surface of the skin.
Telangiectasis appear as tiny,
red, spider-like veins.
The disease results from defects
in the ataxia telangiectasia
mutated (ATM) gene.
Defects in this gene can lead
to abnormal cell death in
various places of the body,
including the part of the brain
that helps coordinate
movement.
